This is a competing renewal application for a project that has been supported by NIH since 2006. In this renewal application, we seek new methods for rapid formation of vascular networks that are relevant to tissue engineering. Our two laboratories have significant experience with the design of biomaterials for protein delivery/tissue engineering and the use of genetic engineering to enhance vascular cell survival and blood vessel formation in vivo: as a result of this collaboration, we have produced a new method, which we call vessel self assembly, that is now ready to be applied to a significant and difficult problem, tissue engineering of liver. Over the past 4 years we have optimized systems in which primary human endothelial cells (ECs), suspended in protein gels, self assemble into vascular conduits in vitro. These self assembled conduits provide perfusion to tissue engineered grafts in vivo after implantation into immunodeficient mouse hosts. We have shown that vessel self assembly can be enhanced by incorporating sustained delivery of pro-angiogenic proteins that act on ECs. We have also shown that the progression to functionally mature vessels is enhanced when ECs are co-implanted with human aortic smooth muscle cells or pericytes (PCs). In this renewal application, we propose, first, to improve the generation of vascular networks via self-assembly from isolated cells and, second, to apply this methodology to tissue engineering by co-transplantation of differentiated epithelial cells within the EC/PC gel constructs with the goal of producing a new, perfused functional tissue. To accomplish these goals we have identified three specific aims for this five-year project. In Aim 1, we seek to improve EC function by identifying and engaging critical pathways mediated by Bcl-2. To accomplish this, we will identify the molecular mechanisms by which Bcl-2 expression enhances vascular self-assembly using a high-throughput, in vitro system (involving cell spheroid suspension in protein gels) that reveals an effect of Bcl-2 on EC tube formation. In Aim 2, we will improve vascular self-assembly by identifying and activating critical functions mediated by PCs. We will test two hypotheses related to optimizing PC effects: a) that PCs can be recruited to EC tubes more effectively if the ECs are genetically altered to inducibly over-express autotaxin, the enzyme needed for releasing lysophophatidic acid, incorporating sustained release of an autotaxin-inducing molecule into our gel system; b) that PCs exert some or all of their maturing effect on ECs by paracrine release of angiopoietin 1. In Aim 3, we will apply the approaches we have already developed- and new approaches as they are discovered in Aims 1 and 2-to use vascular self-assembly to create tissues by transplanting EC/PC/hepatocytes co-cultures for regeneration of liver function. Here, we will use our sustained release systems for maintaining differentiated functions of hepatocytes. In all of our approaches, we rely on materials that are already acceptable to the FDA in clinical settings; therefore, our results in animal models will be ready for translation into clinical practice.